A Liquid Formulation of Alpha-Amylase

ABSTRACT

The present disclosure relates to liquid enzyme formulations containing one or more alpha-amylases for use in starch processing, wherein the pH of the enzyme formulation is about pH 6.0-8.0, and methods of use thereof. The present disclosure further relates to methods of making a liquid enzyme formulation containing one or more alpha-amylase having improved stability, comprising titrating the pH of the liquid enzyme formulation to a range of pH 6.0-8.0.

REFERENCE TO SEQUENCE LISTING SUBMITTED VIA EFS-WEB

This application includes an amino acid sequence listing in computerreadable from (CRF) and conforming to the requirements of 37 C.F.R.1.821 through 1.825. The sequence listing of this application is beingsubmitted to the USPTO via the EFS-WEB server as authorized and setforth in MPEP § 502.05. The sequence listing of this application isfiled in an ASC II text (.txt) file as identified below and is herebyincorporated by reference into the specification of this application inits entirety and for all purposes.

Date of File Name Creation Size (bytes) SEQUENCELISTING_160035WO01 Dec.16, 2016 51.5 KB (52,743 bytes)

BACKGROUND

The present disclosure relates to liquid enzyme formulations containingone or more alpha-amylases. Alpha-amylases are useful in a variety ofindustrial applications such as starch processing, production of a foodproduct, a biofuel, a cleaning product, an animal feed, or a paper pulp,and enhanced oil recovery. The present disclosure further relates tomethods of making and using a liquid enzyme formulation containing oneor more enzymes, such as an alpha-amylase, a protease, a cellulase, aglucoamylase having improved stability.

Currently, some alpha-amylase formulations maintain the enzyme at a highpH (pH 10.7) environment. Alpha-amylases in high pH formulation may havea lowered stability at this high pH range, which can result in ashortened product shelf life. In addition, there are few stable,compatible, and regulatory approved buffering agents for a high pHformulation. Without a buffer, pH of the alpha-amylase formulation canbe unstable, and may drift lower over time. Further, there is potentialof microbial growth due to the lack of a functioning preservativebecause food grade preservatives do not work quite well in this pHrange. While the high pH initially has some anti-microbial effect, thepH eventually drifts down. The formulation may also be incompatible withmost of the common formulations of other enzyme products, which areusually in neutral or slightly acidic pH range. Sometimes it isadvantageous to mix two alpha-amylases of different properties duringstarch hydrolysis. For example, mixing high temperature activealpha-amylase with a low temperature active alpha-amylase may increasethe break down the starch into oligo saccharides and lower the viscosityof the feed stock. Due to the extreme pH difference of different enzymeformulations, it is difficult to blend an alpha-amylase with a high pHformulation with different enzyme(s) having a low pH formulation.

SUMMARY

Some embodiments disclosed herein provide liquid enzyme formulationscomprising: (a) an alpha-amylase; (b) a buffering agent; (c) astabilizer; and (d) a preservative, wherein the pH of the enzymeformulation is about pH 6.0-8.0.

In some embodiments, the pH of the liquid enzyme formulation is about pH6.3-6.7. In some embodiments, the stabilizer comprises sucrose,sorbitol, mannitol, glycerol, trehalose, sodium chloride, sodiumsulfate, or any combination thereof. In some embodiments, the stabilizeris glycerol. In some embodiments, the glycerol is at a concentration ofabout 30% to about 75%. In some embodiments, the glycerol is at aconcentration of about 30% to about 70%. In some embodiments, theglycerol is at a concentration of about 30% to about 65%. In someembodiments, the glycerol is at a concentration of about 30% to about50%. In some embodiments, the glycerol is at a concentration of about38% to about 42%. In some embodiments, the buffering agent comprisessodium citrate, potassium citrate, citric acid, sodium acetate, aceticacid, sodium phosphate, potassium phosphate, or any combination thereof.In some embodiments, the buffering agent comprises sodium citrate. Insome embodiments, sodium citrate is at a concentration of about 0.1% toabout 2.0%. In some embodiments, sodium citrate is at a concentration ofabout 0.1% to about 0.6%. In some embodiments, the alpha-amylase retainsat least 90% of its activity at a temperature of 4-40° C. In someembodiments, the alpha-amylase retains at least 90% of its activity at atemperature of 25-30° C. In some embodiments, the alpha-amylase retainsat least 90% of its activity for 1 year. In some embodiments, thealpha-amylase has a shelf life of at least 1 year. In some embodiments,the alpha-amylase has a shelf life of at least 1 year at 25° C. In someembodiments, the preservative comprises: potassium sorbate, sodiumsorbate, sorbic acid, sodium benzoate, benzoic acid, methyl paraben,calcium propionate, sodium propionate, ammonium propionate, propionicacid, or any combination thereof. In some embodiments, the liquid enzymeformulation comprises at least two preservatives. In some embodiments,the at least two preservatives comprise methyl paraben and potassiumsorbate. In some embodiments, the methyl paraben is at a concentrationof about 0.0% to about 0.3%. In some embodiments, the methyl paraben isat a concentration of about 0.1%. In some embodiments, the potassiumsorbate is at a concentration of about 0.1% to about 0.5%. In someembodiments, the potassium sorbate is at a concentration of about 0.2%.In some embodiments, liquid enzyme formulations comprising: (a) analpha-amylase having an amino acid sequence that is at least 80%identical to SEQ ID NO:1; (b) a sodium citrate; (c) a glycerol; and (d)a potassium sorbate, wherein the pH of the enzyme formulation is aboutpH 6.3-6.7. In some embodiments, liquid enzyme formulations comprising:(a) an alpha-amylase having an amino acid sequence that is at least 80%identical to SEQ ID NO:1; (b) a sodium citrate and a citric acid; (c) aglycerol; and (d) a potassium sorbate and a methyl paraben, wherein thepH of the enzyme formulation is about pH 6.3-6.7.

In some embodiments, the alpha-amylase comprises an amino acid sequencehaving at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identityto any one of the amino acid sequences as set forth in: SEQ ID NO:1, SEQID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ IDNO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ IDNO:12, SEQ ID NO:13, and SEQ ID NO:14.

In some embodiments, the liquid enzyme formulation comprises a secondenzyme. In some embodiments, the second enzyme is selected from thegroup consisting of an alpha-amylase, a beta-amylase, a glucoamylase, aprotease, a phytase, a pullulanase, a cellulase, a cellobiohydrolase, abeta-glucosidase, an endoglucanase, a mannanase, and any combinationthereof.

Some embodiments disclosed herein provide methods of making a liquidenzyme formulation comprising: (a) providing a composition comprising analpha-amylase, wherein the composition has a pH value of about 8.0 toabout 10.5; (b) adding a stabilizer to the composition comprising thealpha-amylase to obtain a liquid enzyme formulation; and (c) titratingthe pH of the liquid enzyme formulation to a range of pH 6.0-8.0.

In some embodiments, step (c) comprises titrating the pH of the liquidenzyme formulation to a range of pH 6.3-6.7. In some embodiments, thetitrating step comprises adding a buffered sodium citrate stock solutionto the liquid enzyme formulation. In some embodiments, the bufferedsodium citrate stock solution is added to a final concentration of0.1-2.0%. In some embodiments, the buffered sodium citrate stocksolution is added to a final concentration of 0.1-0.6%. In someembodiments, the stabilizer comprises glycerol. In some embodiments, theglycerol is added to a final concentration of 30-75% In someembodiments, the glycerol is added to a final concentration of 30-50%.In some embodiments, the glycerol is added to a final concentration of38-42%.

Some embodiments disclosed herein provide uses of a liquid enzymeformulation, comprising: (a) providing a liquid enzyme formulationcomprising an alpha-amylase, a buffering agent, a stabilizer, and apreservative, wherein the pH value of the liquid enzyme formulation isabout pH 6.0-8.0; (b) providing a starch; and (c) contacting the liquidenzymatic formulation with the starch, thereby hydrolyzing the starch.

In some embodiments, the pH of the liquid enzyme formulation is about pH6.3-6.7. In some embodiments, the liquid enzyme formulation is used forthe production of a food ingredient, a food product, a fuel, an alcohol,a cleaning product, such as a detergent, a personal care product, suchas a cosmetic, an animal feed, a paper, a paper pulp, a paper product,or a starch based product. In some embodiments, the liquid enzymeformulation is used for oil and gas well operations, in anotherembodiment the oil and gas well operation is enhanced oil recovery.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an exemplary flowchart illustrating a non-limitingembodiment of the formulation process disclosed herein.

FIG. 2 shows enzyme stability of a high pH alpha-amylase enzymeformulation containing methyl paraben (three different production lots)at six months. The alpha-amylase enzyme formulation contains 40%glycerol, 0.1% methyl paraben, and pH 10.7. The samples were stored at5° C., 22° C., 30° C. and 37° C., respectively.

FIG. 3 shows enzyme stability of a high pH alpha-amylase enzymeformulation having no methyl paraben (three different production lots)at six months. The alpha-amylase enzyme formulation contains 40%glycerol, and pH 10.7. Various samples were stored at 5° C., 22° C., 30°C. and 37° C., respectively.

FIGS. 4A, 4B, & 4C show enzyme stability of an exemplary low pHalpha-amylase (SEQ ID NO:1) enzyme formulation (three differentproduction lots) at six months. The alpha-amylase enzyme formulationcontains 40% glycerol, 0.2% potassium sorbate, 0.1% methyl paraben, pH6.5, and sodium citrate at 5 mM, 10 mM or 20 mM. The samples were storedat 4° C., 25° C. and 40° C., respectively.

FIGS. 5A & 5C show the comparison of enzyme stability betweenalpha-amylase enzyme formulations at two different pH (three differentproduction lots) at six months. The high pH alpha-amylase enzymeformulation contains 40% glycerol, 0.1% methyl paraben, and pH 10.7; thelow pH alpha-amylase enzyme formulation contains 40% glycerol, 0.1%methyl paraben, 0.2% potassium sorbate, 10 mM sodium citrate, pH 6.5.Each of three enzyme production lots were formulated into the twodifferent formulations. The samples were stored at 4° C., 25° C., 30° C.and 40° C., respectively.

FIG. 6 shows the comparison of enzyme stability between alpha-amylaseenzyme formulations at two different pH at twelve months. The high pHalpha-amylase enzyme formulation contains 40% glycerol, 0.1% methylparaben, and pH 10.7; the low pH alpha-amylase enzyme formulationcontains 40% glycerol, 0.1% methyl paraben, 0.2% potassium sorbate, 10mM sodium citrate, pH 6.5. The same enzyme production lot was formulatedinto the two different formulations. The samples were stored at 4° C.,25° C., 30° C. and 40° C., respectively.

FIGS. 7A & 7C show the comparison of enzyme stability between twodifferent alpha-amylase enzyme formulations at high pH (three differentproduction lots) at five months. Formulation A contains 40% glycerol,0.1% methyl paraben, 0.2% potassium sorbate, 10 mM sodium citrate, pH6.5; formulation B contains 50% glycerol, 0.2% potassium sorbate, 10 mMsodium citrate, pH 6.5. Each of three enzyme production lots wereformulated into the two different formulations. The samples were storedat 4° C., 25° C., 30° C. and 40° C., respectively.

DETAILED DESCRIPTION

The current alpha-amylase high pH formulation comprises 40% w/vglycerol, 0.1% w/v methyl paraben, pH 10.7. The selection of pH 10.7 wasthe due to limited solubility of the enzyme at lower pH and the processdifficulty in titrating the pH down across a wide range after downstreamrecovery. The high pH formulation does not provide the necessary productstability because it is unbuffered, pH drift which occurs over timeposes a risk for subsequent microbial contamination and commerciallosses during bulk shipment, warehouse storage, or product storage atindustrial plants. The present disclosure provides a liquid enzymeformulation comprising an alpha-amylase formulation that has a neutralor acidic pH for increased stability and shelf life.

Definitions

The term “liquid enzyme formulation” means to a liquid compositioncomprising an enzyme. In some embodiments, a liquid enzyme formulationfurther comprises a buffer, a stabilizer, and a preservative. In someembodiments, the enzyme is an alpha-amylase, a beta-amylase, aglucoamylase, a protease, a phytase, a pullulanase, a cellulase,cellobiohydrolase, a beta-glucosidase, a endoglucanase, a mannanase, axylanase, or any combination of enzymes thereof.

An enzyme is a biological molecule comprising a sequence of amino acids,wherein the enzyme can catalyze a reaction. Enzyme names are known tothose skilled in the art based on the recommendations of theNomenclature Committee of the International Union of Biochemistry andMolecular Biology (IUBMB). Enzyme names include: an EC (EnzymeCommission) number, recommended name, alternative names (if any),catalytic activity, and other factors. Enzymes are also refereed to as apolypeptide, a protein, a peptide, an amino acid sequence, or isidentified by a SEQ ID NO. in this disclosure.

An enzyme is “thermostable” if it retains a substantial amount of itsactivity after a high temperature treatment of at least about 65° C. toabout 95° C.; or at a temperature greater than 95° C. In someembodiments, the thermostable enzyme retains at least: 5%, 6%, 7%, 8%,9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%,23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%,37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%,51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%,65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%,79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of its enzymatic activity.

Starch can be any virgin, modified, or degraded starch, orpolysaccharide/oligosaccharide. Virgin starches may consist of amylose,amylopectin, or mixtures thereof. Modified starches may compriseoxidized starch, starch esters, or starch ethers.

Types of starch contemplated include virgin starches such as potatostarch; wheat starch, corn starch, rice starch, or other grain basedstarch; or tapioca starch, preferably corn starch. The starches couldalso be any biomass-containing starch components from plants oragricultural residues. Chemically modified starches may also be used,such as hydroxyethyl or hydroxypropyl starches, or else starches whichcontain anionic groups, such as phosphate starch, or else cationizedstarches containing quaternary ammonium groups, preference being givento a degree of substitution DS of 0.01 to 0.2. This degree ofsubstitution DS indicates the number of cationic groups present onaverage in the starch per glucose unit. Particularly preferred areamphoteric starches, which contain not only quaternary ammonium groupsbut also anionic groups such as carboxylate and/or phosphate groups, andwhich may optionally also have undergone chemical modification, havingfor example been hydroxyalkylated or alkyl-esterified. The starches maybe used individually or else in any desired mixtures with one another.

The starches may be present with other hemicelluloses orpolysaccharides, such as but not limited to galactomannans, xylans,arabinoxylans, glucuronoxylans, glucomannans, xyloglucans (such asTamarind seed flour), pectins/pectate, galactans, arabinogalactans. Inthe case where starch used contains other polysaccharides, it isoptional that hemicellulase enzymes or polysaccharide enzymes may beused in combination with the amylase for the preparations of surfacestarch composition of the present disclosure. In one option, xylanases(for example: Luminase™ PB-100, Luminase™ PB-200) from BASF can be used.In fact hemicellulases, xylanases, cellulases, and other enzymes fromany suppler is implied, including from Novozymes, Dyadic, Dupont, andBASF. If the starchy materials contain proteins and fats, protease andlipases may optionally be used in combination with the aforementionedenzymes comprising amylase.

The starch used in the present disclosure, preferably, is a nativestarch, or a substantially un-modified starch. One example is the Pearlstarch from Tate and Lyle. However, the starch used can also be anymodified or partially modified starches, as modified by thermaltreatment, by thermal-mechanical treatment, by acid hydrolysis, byoxidations, by ester derivatizations, (such as starch acetates, starchphosphates), by ether modifications or hydroxyl-alkyl derivatizations(such as hydroxypropyl starches, hydroxyethyl starches or ethylatedstarches, hydroxypropyl starch phosphates, carboxymethyl starches,various cationic starches, and previously enzyme modified starches, andpre-gelatinized starches. Common examples include industrial starchesfrom A. E. Staley, Penford (Ingredion), Tate and Lyle, ADM, Cargill,Rasio, Roguette, and Amylum, to name a few.

As used herein, “carbohydrates,” “saccharide” or “sugar” refers to amacromolecule consisting of carbon (C), hydrogen (H), and oxygen (O)atoms, usually with a hydrogen: oxygen atom ratio of 2:1 (as in water);in other words, with the empirical formula Cm(H2O)n (where m could bedifferent from n). Polysaccharides can have more than one saccharide andare used for the storage of energy. Monosaccharides contain only onesaccharide unit, while a disaccharide can contain two saccharide units,or two joined monosaccharides.

A “buffering agent” means a weak acid or base used to maintain theacidity (pH) of a solution near a chosen value after the addition ofanother acid or base.

A “stabilizer” means a chemical that minimize the instability of anenzyme and therefore maintain its stability. The instability of anenzyme can be due conformational instability, colloidal instability orchemical degradation, which can leads to the loss of enzymatic activity.Examples of stabilizers include but not limited sugars, polyols andsalts.

A “preservative” is an agent that kills microorganisms or inhibits theirgrowth, including disinfectants, antiseptics, and antibiotics, etc.

The term “comprising” as used herein is synonymous with “including,”“containing,” or “characterized by,” and is inclusive or open-ended,which means that additional elements not recited or method steps notrecited can be within the scope of this disclosure.

It is understood that aspects and embodiments of the invention describedherein include “consisting” and/or “consisting essentially of” aspectsand embodiments.

Throughout this disclosure, various aspects are presented in a rangeformat. It should be understood that the description in range format ismerely for convenience and brevity and should not be construed as aninflexible limitation on the scope of the disclosure. Accordingly, thedescription of a range should be considered to have specificallydisclosed all the possible sub-ranges as well as individual numericalvalues within that range. For example, description of a range such asfrom 1 to 6 should be considered to have specifically disclosedsub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4,from 2 to 6, from 3 to 6 etc., as well as individual numbers within thatrange, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of thebreadth of the range.

Other objects, advantages and features of the present disclosure willbecome apparent from the following specification taken in conjunctionwith the accompanying drawings.

In the following description, numerous specific details are set forth toprovide a more thorough understanding of the present disclosure.However, it will be apparent to one of skill in the art that the methodsof the present disclosure may be practiced without one or more of thesespecific details. In other instances, well-known features and procedureswell known to those skilled in the art have not been described in orderto avoid obscuring the disclosure.

Liquid Enzyme Formulations

Some embodiments disclosed herein provide liquid enzyme formulationscomprising an alpha-amylase and a buffering agent, wherein the pH of theenzyme formulation is about pH 6.0-8.0. In some embodiments, the liquidenzyme formulations comprise a stabilizer. In some embodiments, theliquid enzyme formulations comprise a preservative.

An enzyme is a biological molecule comprising a sequence of amino acids,wherein the enzyme can catalyze a reaction. Enzyme names are known tothose skilled in the art based on the recommendations of theNomenclature Committee of the International Union of Biochemistry andMolecular Biology (IUBMB). Enzyme names include: an EC (EnzymeCommission) number, recommended name, alternative names (if any),catalytic activity, and other factors. Enzymes are also known as apolypeptide, a protein, a peptide, an amino acid sequence, or isidentified by a SEQ ID NO. In this disclosure, the alternative names forenzyme can be used interchangeably. An “alpha-amylase” is an enzyme thatcatalyzes the hydrolysis of 1,4-alpha-D-glucosidic linkages to degradepolysaccharides, oligosaccharides, and/or starch into glucose subunits.The enzyme classification for an alpha-amylase is EC 3.2.1.1.

The liquid enzyme formulations disclosed herein may be a pH that issuitable for the stability and maintains the activity of the enzymecontained therein. For example, the pH of the liquid enzyme formulationmay be a value that is, is about, is less than, pH 6.0, pH 6.1, pH 6.2,pH 6.3, pH 6.4, pH 6.5, pH 6.6, pH 6.7, pH 6.8, pH 6.9, pH 7.0, pH 7.1,pH 7.2, pH 7.3, pH 7.4, pH 7.5, pH 7.6, pH 7.7, pH 7.8, pH 7.9, pH 8.0,or a range that is between any two of the above mentioned values. Insome embodiments, the pH of the liquid enzyme formulation is about pH6.0-7.5, pH 6.0-7.0, pH 6.0-6.7, or pH 6.3-6.7.

A variety of suitable buffering agents may be used to adjust the pH ofthe liquid enzyme formulations disclosed herein. For example, the liquidenzyme formulations may comprise a buffering agent selected from thegroup consisting of: sodium citrate, potassium citrate, citric acid,sodium acetate, acetic acid, sodium phosphate, potassium phosphate, andany combination thereof. In some embodiments, the liquid enzymeformulations comprise a buffering agent that is sodium citrate. Thesodium citrate may be included in the liquid enzyme formulations at aconcentration that is, is about, is less than, is more than, 0.1% (w/v),0.2% (w/v), 0.3% (w/v), 0.4% (w/v), 0.5% (w/v), 0.6% (w/v), 0.7% (w/v),0.8% (w/v), 0.9% (w/v), 1.0% (w/v), 1.1% (w/v), 1.2% (w/v), 1.3% (w/v),1.4% (w/v), 1.5% (w/v), 1.6% (w/v), 1.7% (w/v), 1.8% (w/v), 1.9% (w/v),2.0% (w/v), or a range that is between two of any of the above mentionedvalues. In some embodiments, the sodium citrate is at a concentration of0.1-2.0% (w/v) in the liquid enzyme formulation. In some embodiments,the sodium citrate is at a concentration of 0.1-0.6% (w/v) in the liquidenzyme formulation.

A variety of suitable stabilizers may be included in the liquid enzymeformulations disclosed herein. For example, the liquid enzymeformulations may comprise a stabilizer such as sucrose, sorbitol,mannitol, glycerol, trehalose, sodium chloride, sodium sulfate, or anycombination thereof. In some embodiments, the liquid enzyme formulationscomprise a stabilizer that is glycerol. The glycerol may be included inthe liquid enzyme formulations at a concentration that is, is about, isless than, is more than, 30% (w/v), 31% (w/v), 32% (w/v), 33% (w/v), 34%(w/v), 35% (w/v), 36% (w/v), 37% (w/v), 38% (w/v), 39% (w/v), 40% (w/v),41% (w/v), 42% (w/v), 43% (w/v), 44% (w/v), 45% (w/v), 46% (w/v), 47%(w/v), 48% (w/v), 49% (w/v), 50% (w/v), 51% (w/v), 52% (w/v), 53% (w/v),54% (w/v), 55% (w/v), 56% (w/v), 57% (w/v), 58% (w/v), 59% (w/v), 60%(w/v), 61% (w/v), 62% (w/v), 63% (w/v), 64% (w/v), 65% (w/v), 66% (w/v),67% (w/v), 68% (w/v), 69% (w/v), 70% (w/v), 71% (w/v), 72% (w/v), 73%(w/v), 74% (w/v), 75% (w/v), or a range that is between two of any ofthe above mentioned values. In some embodiments, the glycerol is at aconcentration of 30-75% (w/v) in the liquid enzyme formulation. In someembodiments, the glycerol is at a concentration of 30-70% (w/v) in theliquid enzyme formulation. In some embodiments, the glycerol is at aconcentration of 30-65% (w/v) in the liquid enzyme formulation. In someembodiments, the glycerol is at a concentration of 30-50% (w/v) in theliquid enzyme formulation. In some embodiments, the glycerol is at aconcentration of 38-42% (w/v) in the liquid enzyme formulation.

The liquid enzyme formulations disclosed herein have improved stabilitycharacteristics. For example, the alpha-amylase of the liquid enzymeformulation disclosed herein can retain its activity at a highpercentage, at elevated temperatures, for a long period of time, andthus the liquid enzyme formulation has a longer shelf life. “Shelf life”as used herein refers to how long the liquid enzyme product can bestored at a particular temperature before it begins to lose itsenzymatic activity. In some embodiments, the alpha-amylase retains atleast 50%, 60%, 70%, 80%, 90%, 95%, or more, of its activity at atemperature, such as 4-40° C. In some embodiments, the alpha-amylaseretains at least 50%, 60%, 70%, 80%, 90%, 95%, or more, of its activityat a temperature of 25-30° C. In some embodiments, the alpha-amylaseretains at least 50%, 60%, 70%, 80%, 90%, 95%, or more, of its activityfor 1 year. In some embodiments, the alpha-amylase has a shelf life ofat least 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8months, 9 months, 10 months, 11 months, 1 year, or more. In someembodiments, the alpha-amylase has a shelf life of at least 2 months, 3months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10months, 11 months, 1 year, or more, at 25° C. In some embodiments, thealpha-amylase has a shelf life of at least 2 months, 3 months, 4 months,5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months,1 year, or more, at 37° C.

As disclosed herein, the liquid enzyme formulations may comprise avariety of preservatives, such as potassium sorbate, sodium sorbate,sorbic acid, sodium benzoate, benzoic acid, methyl paraben, calciumpropionate, sodium propionate, ammonium propionate, propionic acid, orany combination thereof. In some embodiments, the liquid enzymeformulations disclosed herein comprise no preservative. In someembodiments, the liquid enzyme formulations disclosed herein compriseonly one preservative, such as potassium sorbate. In some embodiments,the liquid enzyme formulations disclosed herein comprise at least twopreservatives, such as methyl paraben and potassium sorbate. Methylparaben may be included in the liquid enzyme formulations at aconcentration that is, is about, is less than, is more than, 0.00%(w/v), 0.01% (w/v), 0.02% (w/v), 0.03% (w/v), 0.04% (w/v), 0.05% (w/v),0.06% (w/v), 0.07% (w/v), 0.08% (w/v), 0.09% (w/v), 0.1% (w/v), 0.2%(w/v), 0.3% (w/v), or a range that is between two of any of the abovementioned values. In some embodiments, the methyl paraben is at aconcentration of 0.05-0.3% (w/v) in the liquid enzyme formulation. Insome embodiments, the methyl paraben is at a concentration of 0.1% (w/v)in the liquid enzyme formulation. The potassium sorbate may be includedin the liquid enzyme formulations at a concentration that is, is about,is less than, is more than, 0.1% (w/v), 0.2% (w/v), 0.3% (w/v), 0.4%(w/v), 0.5% (w/v), or a range that is between two of any of the abovementioned values. In some embodiments, the potassium sorbate is at aconcentration of 0.1-0.5% (w/v) in the liquid enzyme formulation. Insome embodiments, the potassium sorbate is at a concentration of 0.2%(w/v) in the liquid enzyme formulation.

In some embodiments, the liquid enzyme formulations may comprise sodiumcitrate at a concentration of 0.1-2.0% (w/v), glycerol at aconcentration of 30-75% (w/v), methyl paraben at a concentration of0.0-0.3% (w/v), and potassium sorbate at a concentration of 0.1-0.5%(w/v), in the liquid enzyme formulation. In some embodiments, the liquidenzyme formulations may comprise sodium citrate at a concentration of0.1-0.6% (w/v), glycerol at a concentration of 38-50% (w/v), methylparaben at a concentration of 0.1% (w/v), and potassium sorbate at aconcentration of 0.2% (w/v), in the liquid enzyme formulation.

Some embodiments disclosed herein provide polypeptides having amylaseactivities and their uses in the production of a food product, abiofuel, a cleaning product, an animal feed, or a paper pulp, etc., andenhanced oil recovery, etc. In some embodiments, the amylase can be athermostable alpha amylase, for example a thermostable alpha amylasefrom bacteria (e.g., Bacillus) or fungi, or any combination (mixture) ofenzymes thereof.

In some embodiments, the alpha amylase comprises or has an amino acidsequence set forth in one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ IDNO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, and SEQ IDNO:14.

In some embodiments, the alpha amylase is variant of the parent alphaamylase that comprises or has an amino acid sequence at least 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence setforth in any of the following amino acid sequences: SEQ ID NO:1, SEQ IDNO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7,SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQID NO:13, and SEQ ID NO:14.

In some embodiments, the alpha amylase is one of the alpha amylasedisclosed in the following patent applications: WO2014007921,WO2011017093, WO2010074999, WO1994019454, WO2010008841, WO2011080352,WO2011080354, WO2011082429, WO199744361, WO2002068589, WO202068597,WO2002092802, WO2003083054, WO2004091544, WO2009020459, WO2013116175,WO2013148163, WO2008080093, and WO2003018766. The contents of thesepatent applications are hereby incorporated by reference in theirentireties.

In some embodiments, the alpha amylase is FUELZYME™ (BASF Enzymes LLC,San Diego, Calif.). Other non-limiting examples of the amylases suitablefor use in the methods and compositions disclosed herein include:LpHera® from Novozymes; SPEZYME®XTRA, SPEZYME® CL, SPEZYME® Alpha,SPEZYME® RSL, SPEZYME® FRED, SPEZYME® LT 300, AmyS, AmyL, BAN®480L,Liquozyme® Supra, Liquozyme® SCDS, MAX-LIFE™ P100, Maltogenase L,CLARASE® L, Liquozyme® SC, Termamyl® SC, Veretase, Liqozyme® SC4x,Liquozyme® Supra 2.8, Liquozyme® supra 2, Liquozyme® X, Termamyl® 120L,SPEZYME® ALPHA, Clearflow® AA, Optitherm™ and Takatherm™, Keistase™,Avantec.

In addition to the alpha amylase, the liquid enzyme formulationsdisclosed herein can include one or more additional enzymes. In someembodiments, the liquid enzyme formulation comprises a second enzyme. Insome embodiments, the second enzyme is selected from the groupconsisting of an alpha-amylase, a beta-amylase, a glucoamylase, aprotease, a phytase, a pullulanase, a cellulase, a cellobiohydrolase, abeta-glucosidase, an endoglucanase, a mannanase, and any combinationthereof. In some embodiments, the liquid enzyme formulation comprisestwo or more enzymes. For example, the liquid enzyme formulation cancontain the alpha amylase, a second enzyme, and a third enzyme. Thethird enzyme can be, for example, an alpha-amylase, a beta-amylase, aglucoamylase, a protease, a phytase, a pullulanase, a cellulase, acellobiohydrolase, a beta-glucosidase, an endoglucanase, a mannanase, orany combination thereof.

In some embodiments, the liquid enzyme formulations comprise a cellulaseor a variant thereof. In some embodiments, the cellulase is any of thecellulases derived from hyperthermophilic bacteria and/or non-naturallyoccurring variants thereof described in PCT publication WO 2009/020459(the entire disclosure of which is incorporated herein by reference).

In some embodiments, the liquid enzyme formulations comprise abeta-glucosidase or a variant thereof. In some embodiments, thebeta-glucosidase can be a commercially available product, or any mixturethereof. In some embodiments, the beta-glucosidase can be any of thebeta-glucosidases from Thermotoga maritima (BGT), Phanerochaetechrysosporium (BGP), or Aspergillus niger (BG) (Sigma, St. Louis, Mo.).

Methods of Making a Liquid Enzyme Formulation

Some embodiments disclosed herein provide methods of making a liquidenzyme formulation, wherein the methods comprise titrating the pH of theliquid enzyme formulation to a range of pH 6.0-8.0. In some embodiments,the methods comprise a step of providing a composition comprising analpha-amylase, wherein the composition has a pH of about pH 8.0-10.5. Insome embodiments, the methods comprise a step of adding a stabilizer tothe composition comprising alpha-amylase to obtain a liquid enzymeformulation.

A non-limiting example of the method 100 of making a liquid enzymeformulation in accordance with the embodiments disclosed herein isillustrated in the flow diagram shown in FIG. 1. As illustrated in FIG.1, the method 100 can include one or more functions, operations oractions as illustrated by one or more operations 110-160.

Method 100 can begin at optional operation 110, “Obtaining recoverymaterial.” Recovery, often also referred to as downstream recovery, is aprocess of separating enzyme in the fermentation broth from theexpression organism, and the enzyme is potentially further concentrated,purified, refined, washed or buffered exchanged in the solution.Recovery material, sometimes also referred to as enzyme concentrate,contains enzyme in concentrated solution after the recovery process butbefore the formulation steps. Operation 110 can be followed by operation120, “Pre-formulation.” In this step, a stabilizer may be added to therecovery material, to initially stabilize the enzyme, prior to theadditions of other formulation ingredients and additional adjustments tothe final formulated product. Operation 120 can be followed by optionaloperation 130, “Adding preservative to pre-formulation mixture.”Operation 130 can be followed by optional operation 140, “Small volumepH titration test.” In this test, a stock solution of concentratedacidic buffer at a particular pH is added and mixed to thepre-formulation mixture incrementally, and the pH of the solution ismeasured. When the pH of the solution reaches the target range of thefinal formulation, the volume of the stock buffer solution required isrecorded. An additional buffer stock solution at a lower pH may berequired to repeat the titration test if too much buffer stock volume isused in the initial test. The purpose of this titration test is todetermine the precise pH and volume of an acidic buffer stock solutionrequired so that the final formulation solution reaches target pH rangewithin the concentration range of the buffer reagent in the finalformulation. In some embodiments, no additional strong acid is used forthe pH titration. In some embodiments, a set volume of the buffer stocksolution is added to the pre-formulation mixture, and then a strong acidis used to adjust the final pH to the formulation target in thetitration test. The exact volumes of the buffer stock solution and acidused are recorded. Operation 140 can be followed by operation 150“Titrating pH of liquid enzyme formulation.” This step may be carriedout based on the result from Operation 140. The volume and pHrequirements of the acidic buffer stock solution and the strong acid arescaled up to the entire production volume. Operation 150 can be followedby optional operation 160, “Obtaining final formulated liquid productand packaging.”

In FIG. 1, operations 110-160 are illustrated as being performedsequentially with operation 110 first and operation 160 last. It will beappreciated, however, that these operations can be combined and/ordivided into additional or different operations as appropriate to suitparticular embodiments. For example, additional operations can be addedbefore, during or after one or more operations 110-160. In someembodiments, one or more of the operations can be performed at about thesame time. In some embodiments, the method only consists of operations120 and 150, but not any other operations. In some embodiments, themethod consists essentially of operations 120 and 150. In someembodiments, the method only consists of operations 120, 150 and one ofoperations 110, 130, 140 and 160, but not any other operations. In someembodiments, the method only consists of operations 120, 150 and two ofoperations 110, 130, 140 and 160, but not any other operations. In someembodiments, the method only consists of operations 120, 150 and one ormore of operations 110, 130, 140 and 160, but not any other operations.

At optional operation 110, “Obtaining recovery material,” the recoverymaterial is not particularly limited and can be any compositioncomprising an alpha-amylase. The pH of the recovery material is notparticularly limited. For example, the composition comprising analpha-amylase may have a pH value that is, is about, is less than, ismore than, pH 8.0, pH 8.1, pH 8.2, pH 8.3, pH 8.4, pH 8.5, pH 8.6, pH8.7, pH 8.8, pH 8.9, pH 9.0, pH 9.1, pH 9.2, pH 9.3, pH 9.4, pH 9.5, pH9.6, pH 9.7, pH 9.8, pH 9.9, pH 10.0, pH 10.1, pH 10.2, pH 10.3, pH10.4, pH 10.5, or a range that is between any two of the above mentionedvalues. In some embodiments, the pH of the composition comprising analpha-amylase may be adjusted to pH 10.0 if needed. In some embodiments,the pH of the composition comprising an alpha-amylase is about pH8.0-10.5. In some embodiments, the composition comprising analpha-amylase may have an enzyme activity, e.g., alpha-amylase activitythat is at least 300,000 MWU/g (Modified Wohlgemuth Units). In someembodiments, the composition comprising an alpha-amylase may have anenzyme concentration, e.g., alpha-amylase, that is about 300,000-400,000MWU/g.

At operation 120, “Pre-formulation,” a variety of stabilizers may beadded to the composition comprising an alpha-amylase. For example, astabilizer such as sucrose, sorbitol, mannitol, glycerol, trehalose,sodium chloride, sodium sulfate, or any combination thereof, may beadded to the composition comprising an alpha-amylase. In someembodiments, glycerol is added to the composition comprising analpha-amylase as stabilizer. The glycerol may be added to thecomposition comprising an alpha-amylase to a final concentration thatis, is about, is less than, is more than, 30% (w/v), 31% (w/v), 32%(w/v), 33% (w/v), 34% (w/v), 35% (w/v), 36% (w/v), 37% (w/v), 38% (w/v),39% (w/v), 40% (w/v), 41% (w/v), 42% (w/v), 43% (w/v), 44% (w/v), 45%(w/v), 46% (w/v), 47% (w/v), 48% (w/v), 49% (w/v), 50% (w/v), 51% (w/v),52% (w/v), 53% (w/v), 54% (w/v), 55% (w/v), 56% (w/v), 57% (w/v), 58%(w/v), 59% (w/v), 60% (w/v), 61% (w/v), 62% (w/v), 63% (w/v), 64% (w/v),65% (w/v), 66% (w/v), 67% (w/v), 68% (w/v), 69% (w/v), 70% (w/v), 71%(w/v), 72% (w/v), 73% (w/v), 74% (w/v), 75% (w/v), or a range that isbetween two of any of the above mentioned values. In some embodiments,the glycerol is added to the composition comprising an alpha-amylase toa final concentration of 30-50% (w/v). In some embodiments, the glycerolis added to the composition comprising an alpha-amylase to a finalconcentration of 38-42% (w/v).

At optional operation 130, “Adding preservative to pre-formulationmixture,” a variety of preservatives may be added. A preservative suchas a potassium sorbate, sodium sorbate, a sorbic acid, a sodiumbenzoate, a benzoic acid, a methyl paraben, a calcium propionate, asodium propionate, an ammonium propionate, a propionic acid, or anycombination thereof, may be added to the pre-formulation mixture. Insome embodiments, no preservative is added to the pre-formulationmixture. In some embodiments, one preservative, added to thepre-formulation mixture. In another embodiment, the one preservativeadded to the pre-formulation mixture is potassium sorbate. In someembodiments, at least two preservatives, such as methyl paraben andpotassium sorbate, can be added to the pre-formulation mixture. Themethyl paraben can be added to the pre-formulation mixture to a finalconcentration that is, is about, is less than, is more than, 0.00%(w/v), 0.01% (w/v), 0.02% (w/v), 0.03% (w/v), 0.04% (w/v), 0.05% (w/v),0.06% (w/v), 0.07% (w/v), 0.08% (w/v), 0.09% (w/v), 0.1% (w/v), 0.2%(w/v), 0.3% (w/v), or a range that is between two of any of the abovementioned values. In some embodiments, the methyl paraben may be addedto the pre-formulation mixture to a final concentration of 0.05-0.3%(w/v). In some embodiments, the methyl paraben may be added to thepre-formulation mixture to a final concentration of 0.1% (w/v). Thepotassium sorbate may be added to the pre-formulation mixture to a finalconcentration that is, is about, is less than, is more than, 0.1% (w/v),0.2% (w/v), 0.3% (w/v), 0.4% (w/v), 0.5% (w/v), or a range that isbetween two of any of the above mentioned values. In some embodiments,the potassium sorbate is added to the pre-formulation mixture to a finalconcentration of 0.1-0.5% (w/v). In some embodiments, the potassiumsorbate is added to the pre-formulation mixture to a final concentrationof 0.2% (w/v).

At optional operation 140, “Small volume pH titration test,” the pH ofthe acidic buffer stock solution may be a value that is, is about, isless than, is more than, pH 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5,or a range that is between any two of the above mentioned values. Insome embodiments, the pH of the acidic buffer stock solution is about pH4.0. In other embodiments, the pH of the acidic buffer stock solution isabout pH 3.5. The concentration of the buffer stock solution may be avalue that is, is about, is less than, is more than, 5% (w/v), 10%(w/v), 15% (w/v), 20% (w/v), 25% (w/v), 30% (w/v), 35% (w/v), 40% (w/v),or a range that is between any two of the above mentioned values. Avariety of suitable strong acid may be used to titrate the pH in thesmall volume titration test. For example, the pH during the titrationtest may be titrated using an acid selected from the group consistingof: citric acid, acetic acid, sulfuric acid, phosphoric acid,hydrochloric acid, and any combination thereof. Small volume in thistest refers to a sample volume that is convenient and consistent for labscale titration, solution mixing and pH measurement, for example, 100milliliters to 1 liter.

At operation 150, “Titrating pH of liquid enzyme formulation,” the pH ofthe liquid enzyme formulation can vary. For example, the pH of theliquid enzyme formulation may be a value that is, is about, is lessthan, pH 6.0, pH 6.1, pH 6.2, pH 6.3, pH 6.4, pH 6.5, pH 6.6, pH 6.7, pH6.8, pH 6.9, pH 7.0, pH 7.1, pH 7.2, pH 7.3, pH 7.4, pH 7.5, pH 7.6, pH7.7, pH 7.8, pH 7.9, pH 8.0, or a range that is between any two of theabove mentioned values. In some embodiments, the pH of the liquid enzymeformulation is about pH 6.3-6.7.

A variety of suitable buffering agents may be used to titrate the pH ofthe liquid enzyme formulations disclosed herein. For example, the pH ofthe liquid enzyme formulations may be titrated using a buffering agentselected from the group consisting of: sodium citrate, potassiumcitrate, citric acid, sodium acetate, acetic acid, sodium phosphate,potassium phosphate, and any combination thereof. In preferredembodiments, the pH of the liquid enzyme formulations may be titratedusing a buffering agent that is sodium citrate. The sodium citrate maybe added to the liquid enzyme formulations to a final concentration thatis, is about, is less than, is more than, 0.1% (w/v), 0.2% (w/v), 0.3%(w/v), 0.4% (w/v), 0.5% (w/v), 0.6% (w/v), 0.7% (w/v), 0.8% (w/v), 0.9%(w/v), 1.0% (w/v), 1.1% (w/v), 1.2% (w/v), 1.3% (w/v), 1.4% (w/v), 1.5%(w/v), 1.6% (w/v), 1.7% (w/v), 1.8% (w/v), 1.9% (w/v), 2.0% (w/v), or arange that is between two of any of the above mentioned values. In someembodiments, the sodium citrate may be added to the liquid enzymeformulations to a final concentration of 0.1-2.0% (w/v). In someembodiments, the sodium citrate may be added to the liquid enzymeformulations to a final concentration of 0.1-0.6% (w/v). In someembodiments, a buffered sodium citrate stock solution may be used,instead of a strong acid solution (including citric acid), in order toavoid enzyme precipitation. In some embodiments, the buffered sodiumcitrate stock solution has a pH of about 3.0-5.0.

At optional operation 160, “Obtaining final formulated liquid productand packaging,” the liquid enzyme formulation after pH adjustment isobtained.

Uses of a Liquid Enzyme Formulation

The liquid enzyme formulations disclosed herein can be used in anyapplications in which the alpha amylase is suitable. Some embodimentsdisclosed herein provide uses of a liquid enzyme formulation comprisingan alpha-amylase, a buffering agent, a stabilizer and a preservative,wherein the pH of the liquid enzyme formulation is about pH 6.0-8.0 forhydrolyzing a starch. In some embodiments, the liquid enzyme formulationis used for the production of a food product, a biofuel, a cleaningproduct, an animal feed, or a paper pulp. In some embodiments, theliquid enzyme formulation is used for enhanced oil recovery.

EXAMPLES

The examples which follow illustrate aspects of the present disclosure.The percentages in the examples are by weight, unless otherwise stated.

In order to facilitate understanding, the specific embodiments areprovided to help interpret the technical proposal, that is, theseembodiments are only for illustrative purposes, but not in any way tolimit the scope of the invention. Unless otherwise specified,embodiments do not indicate the specific conditions, are in accordancewith the conventional conditions or the manufacturer's recommendedconditions.

Example 1 Preparation of Buffered Low pH Alpha-Amylase Formulation withStrong Acid Titration

Recovery material representing product which would be formulated(“concentrate”) was obtained and alpha-amylase enzyme activity (MWU/g;MWU, Modified Wohlgemuth Units), pH, and microbial load (CFU/mL; CFU,colony forming units) were determined upon receipt. pH of theconcentrate was adjusted to 10.7 if needed. Activity target for theconcentrate was ≥300,000 MWU/g (necessary to meet pre-formulation targetof 185,000 MWU/g after addition of 40% w/v glycerol).

Pre-formulation mixture was prepared by combining concentrate, 40% w/vglycerol, and DI-water. pH of the pre-formulation mixture was adjustedto 10.7 with 1N NaOH. Activity target at pre-formulation step was185,000 MWU/g.

Pre-formulation mixture was optionally pasteurized by heating at 70° C.for 1 hour.

To prepare a low pH formulation, 100× citrate phosphate buffer stocksolution (326 mM citric acid, 436 mM Na₂HPO₄, 800 mM NaOH, pH 6.5) wasadded to the mixture to 1× final concentration (3.3 mM citric acid, 4.4mM Na₂HPO₄, 8 mM NaOH). Following buffer addition, preservatives wereadded to 0.1% w/v final concentration each (methyl paraben, added from25% w/v stock in 100% ethanol; potassium sorbate, added from 25% w/vstock solution in DI water). Additional glycerol was added to the finalformulation solution to achieve 40% w/v glycerol concentration. Thefinal pH of the formulation mixture was adjusted to 6.5 with 12% citricacid. To prepare a high pH formulation (unbuffered, pH 10.7), 0.1% w/vmethyl paraben was added to the pre-formulation mixture, and pH wasadjusted to 10.7 with 1N NaOH. Additional glycerol was added to thefinal formulation solution to achieve 40% w/v glycerol concentration.Activity target for the final formulation was 160,000 MWU/g.

In some embodiments, the formulation mixture may be aliquoted in sterileplastic containers (bottles or tubes) and stored at designatedtemperatures where stability was monitored (−20° C., 5° C., 22° C., 30°C., and 37° C.).

Enzyme activity was monitored using MWU assay, protein quality andintegrity were monitored using HPLC/Reverse phase and SDS PAGE, pH andmicrobial stability were monitored monthly for minimum of three months.

pH stability was determined by taking an aliquot of formulated materialstored at each designated temperature and determining the pH of thesolution. Microbial stability was determined by taking an aliquot offormulated material stored at each designated temperature and plating200 μl on 20 mm TSA plates (tryptic soy agar, DIFCO Laboratories).

Example 2 Preparation of Buffered Low pH Alpha-Amylase FormulationContaining Methyl Paraben without Strong Acid Titration

Recovery material representing product which would be formulated(“concentrate”) was obtained and enzyme (alpha-amylase) activity (MWU/g;MWU, Modified Wohlgemuth Units), pH, and microbial load (CFU/mL; CFU,colony forming units) were determined upon receipt. Activity target forthe concentrate was ≥300,000 MWU/g (necessary to meet pre-formulationtarget of 185,000 MWU/g after addition of 40% w/v glycerol).

Pre-formulation mixture was prepared by combining concentrate, 40% w/vglycerol, and DI-water. Activity target at pre-formulation step was185,000 MWU/g.

Pre-formulation mixture was optionally pasteurized by heating at 70° C.for 1 hour.

To prepare a low pH formulation, preservatives were added to thepre-formulation mixture to final concentrations of 0.1% w/v for methylparaben (added from 25% w/v stock solution in 100% ethanol or 10% w/vstock solution from mono propylene glycol) and 0.2% potassium sorbate(added from 25% w/v stock solution in DI water). One kilogram of thispre-formulation mixture with preservatives was retrieved. Small volumesof 1 molar sodium citrate buffer stock solution at pH 4.0 were added tothe mixture at 1 gram step increment. The mixture was thoroughly mixedand pH was constantly measured. When the mixture pH reached 6.5, thetitration test was stopped and the total amount of sodium citrate stocksolution added was recorded. Based on the 1-kilogram pH titration test,the amount of buffer stock solution needed for formulating the entireproduction volume was calculated. This volume of 1 molar sodium citratebuffer stock solution at pH 4.0 was then added to the entirepre-formulation mixture with preservatives. Additional glycerol wasadded to the final formulation solution to achieve 40% w/v glycerolconcentration. The pH target of the final formulation mixture was 6.5.If additional pH adjustment was needed, 1 molar sodium citrate bufferstock solution at pH 4.0 or 10% sodium hydroxide would be used. Activitytarget for the final formulation was 160,000 MWU/g.

The formulation mixture was aliquoted in sterile plastic containers(bottles or tubes) and stored at designated temperatures where stabilitywas monitored (−20° C., 4° C., 25° C., and 40° C.).

Enzyme activity was monitored using MWU assay, protein quality andintegrity were monitored using HPLC/Reverse phase and SDS PAGE, pH andmicrobial stability were monitored monthly for minimum of three months.

pH stability was determined by taking an aliquot of formulated materialstored at each designated temperature and determining the pH of thesolution. Microbial stability was determined by taking an aliquot offormulated material stored at each designated temperature and plating200 μl on 20 mm TSA plates (tryptic soy agar, DIFCO Laboratories).

Example 3 Preparation of Buffered Low pH Alpha-Amylase FormulationContaining No Methyl Paraben without Strong Acid Titration

Recovery material representing product which would be formulated(“concentrate”) was obtained and enzyme (alpha-amylase) activity (MWU/g;MWU, Modified Wohlgemuth Units), pH, and microbial load (CFU/mL; CFU,colony forming units) were determined upon receipt. Activity target forthe concentrate was ≥325,000 MWU/g (necessary to meet pre-formulationtarget of 200,000 MWU/g after addition of 40% w/v glycerol).

Pre-formulation mixture was prepared by combining concentrate, 40% w/vglycerol, and DI-water. Activity target at pre-formulation step was200,000 MWU/g.

Pre-formulation mixture was optionally pasteurized by heating at 70° C.for 1 hour.

To prepare a low pH formulation, a preservative was added to thepre-formulation mixture to final concentrations of 0.2% potassiumsorbate (added from 25% w/v stock solution in DI water). One kilogram ofthis pre-formulation mixture with preservative was retrieved. Smallvolumes of 1 molar sodium citrate buffer stock solution at pH 4.0 wereadded to the mixture at 1 gram step increment. The mixture wasthoroughly mixed and pH was constantly measured. When the mixture pHreached 6.5, the titration test was stopped and the total amount ofsodium citrate stock solution added was recorded. Based on the1-kilogram pH titration test, the amount of buffer stock solution neededfor formulating the entire production volume was calculated. This volumeof 1 molar sodium citrate buffer stock solution at pH 4.0 was then addedto the entire pre-formulation mixture with preservative. Additionalglycerol was added to the final formulation solution to achieve 50% w/vglycerol concentration. The pH target of the final formulation mixturewas 6.5. If additional pH adjustment was needed, 1 molar sodium citratebuffer stock solution at pH 4.0 or 10% sodium hydroxide would be used.Activity target for the final formulation was 160,000 MWU/g.

The formulation mixture was aliquoted in sterile plastic containers(bottles or tubes) and stored at designated temperatures where stabilitywas monitored (−20° C., 4° C., 25° C., and 40° C.).

Enzyme activity was monitored using MWU assay, protein quality andintegrity were monitored using HPLC/Reverse phase and SDS PAGE, pH andmicrobial stability were monitored monthly for minimum of three months.

pH stability was determined by taking an aliquot of formulated materialstored at each designated temperature and determining the pH of thesolution. Microbial stability was determined by taking an aliquot offormulated material stored at each designated temperature and plating200 μl on 20 mm TSA plates (tryptic soy agar, DIFCO Laboratories).

What is claimed is:
 1. A liquid enzyme formulation comprising: (a) analpha-amylase; (b) a buffering agent; (c) a stabilizer; and (d) apreservative, wherein the pH of the enzyme formulation is about pH6.0-8.0.
 2. The liquid enzyme formulation of claim 1, wherein the pH ofthe liquid enzyme formulation is about pH 6.3-6.7.
 3. The liquid enzymeformulation of claim 1, wherein the stabilizer comprises sucrose,sorbitol, mannitol, glycerol, trehalose, sodium chloride, sodiumsulfate, or any combination thereof.
 4. The liquid enzyme formulation ofclaim 1, wherein the buffering agent comprises: sodium citrate,potassium citrate, citric acid, sodium acetate, acetic acid, sodiumphosphate, potassium phosphate, or any combination thereof.
 5. Theliquid enzyme formulation of claim 1, wherein the alpha-amylase retainsat least 90% of its activity at a temperature of 4-40° C.
 6. The liquidenzyme formulation of claim 1, wherein the alpha-amylase retains atleast 90% of its activity at a temperature of 25-30° C.
 7. The liquidenzyme formulation of claim 1, wherein the alpha-amylase retains atleast 90% of its activity for 1 year.
 8. The liquid enzyme formulationof claim 1, wherein the alpha-amylase has a shelf life of at least 1year.
 9. The liquid enzyme formulation of claim 8, wherein thealpha-amylase has a shelf life of at least 1 year at 25° C.
 10. Theliquid enzyme formulation of claim 1, wherein the preservativecomprises: potassium sorbate, sodium sorbate, sorbic acid, sodiumbenzoate, benzoic acid, methyl paraben, calcium propionate, sodiumpropionate, ammonium propionate, propionic acid, or any combinationthereof.
 11. The liquid enzyme formulation of claim 1, furthercomprising at least two preservatives.
 12. The liquid enzyme formulationof claim 1, wherein the alpha-amylase comprises an amino acid sequencehaving at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identityto one of the amino acid sequences set forth in SEQ ID NO:1, SEQ IDNO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7,SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQID NO:13, and SEQ ID NO:14.
 13. The liquid enzyme formulation of claim1, further comprising a second enzyme.
 14. The liquid enzyme formulationof claim 13, wherein the second enzyme is selected from the groupconsisting of an alpha-amylase, a beta-amylase, a glucoamylase, aprotease, a phytase, a pullulanase, a cellulase, a cellobiohydrolase, abeta-glucosidase, an endoglucanase, a mannanase, a xylanase, and anycombination thereof.
 15. A method of making a liquid enzyme formulationcomprising: (a) providing a composition comprising an alpha-amylase,wherein the composition has a pH value of about 8.0 to about 10.5; (b)adding a stabilizer to the composition comprising the alpha-amylase toobtain a liquid enzyme formulation; and (c) titrating the pH of theliquid enzyme formulation to a range of pH 6.0-8.0.
 16. Use of a liquidenzyme formulation, comprising: (a) providing a liquid enzymeformulation comprising an alpha-amylase, a buffering agent, astabilizer, and a preservative, wherein the pH value of the liquidenzyme formulation is about pH 6.0-8.0; (b) providing a starch; and (c)contacting the liquid enzymatic formulation with the starch, therebyhydrolyzing the starch.
 17. The use of claim 16, for the production of afood ingredient, a food product, a fuel, an alcohol, a cleaning product,a personal care product, an animal feed, a paper, a paper pulp, a paperproduct, or a starch based product.
 18. The use of claim 16, in oil andgas well operations.
 19. A liquid enzyme formulation comprising: (a) analpha-amylase; (b) a buffering agent; (c) a stabilizer; and wherein thepH of the enzyme formulation is about pH 6.0-8.0.
 20. The liquid enzymeformulation of claim 19, wherein the alpha-amylase comprises an aminoacid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity to one of the amino acid sequences set forth in SEQ IDNO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6,SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQID NO:12, SEQ ID NO:13, and SEQ ID NO:14.
 21. The liquid enzymeformulation of claim 19, wherein the buffering agent comprises: sodiumcitrate, potassium citrate, citric acid, sodium acetate, acetic acid,sodium phosphate, potassium phosphate, or any combination thereof. 22.The liquid enzyme formulation of claim 19, wherein the stabilizercomprises sucrose, sorbitol, mannitol, glycerol, trehalose, sodiumchloride, sodium sulfate, or any combination thereof.
 23. The liquidenzyme formulation of claim 1, wherein the pH of the liquid enzymeformulation is about pH 6.3-6.7.
 24. A liquid enzyme formulationcomprising: (a) an alpha-amylase; wherein the alpha-amylase comprises:an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100% sequence identity to one of the amino acid sequences set forth inSEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ IDNO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11,SEQ ID NO:12, SEQ ID NO:13, and SEQ ID NO:14; (b) a buffering agent;wherein the buffering agent comprises: sodium citrate, potassiumcitrate, citric acid, sodium acetate, acetic acid, sodium phosphate,potassium phosphate, or any combination thereof. (c) a stabilizer,wherein the stabilizer comprises: sucrose, sorbitol, mannitol, glycerol,trehalose, sodium chloride, sodium sulfate, or any combination thereof;and wherein the pH of the enzyme formulation is about pH 6.0-8.0.